CN111693617A - Preparation method of aptamer molecular imprinting synergistic recognition stainless steel mesh - Google Patents
Preparation method of aptamer molecular imprinting synergistic recognition stainless steel mesh Download PDFInfo
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Abstract
The invention relates to a preparation method of a nucleic acid aptamer molecular imprinting synergistic recognition stainless steel mesh, which comprises the following steps: (1) sequentially carrying out organic solvent soaking, alkali washing, deionized water washing and drying treatment on the stainless steel mesh; (2) performing surface treatment on the stainless steel mesh obtained in the step (1) by using tannic acid; (3) performing silanization treatment on the stainless steel mesh obtained in the step (2); (4) and (3) adding the stainless steel mesh, the template molecules and the aptamer obtained in the step (3) into a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, then sequentially adding a functional monomer, a cross-linking agent and an initiator to perform a polymerization reaction, and removing the template molecules to obtain the aptamer molecular imprinting synergistic recognition stainless steel mesh. The invention improves the specificity recognition capability of the ligand template molecule through the aptamer-template molecule-functional monomer cooperative recognition, and realizes the advantage complementation of two molecular recognition technologies of molecular imprinting and aptamer.
Description
Technical Field
The invention relates to the field of material preparation and solid phase extraction, in particular to a preparation method of a nucleic acid aptamer molecular imprinting synergistic recognition stainless steel mesh.
Background
With the development of socioeconomic, people pay more and more attention to the food safety problem. At present, about one fourth of the grain and feed contains mycotoxins, which not only cause direct economic loss to the livestock industry, but also partially cause teratogenicity and carcinogenicity to human body, and possibly enter the human body through food and slowly accumulate in the human body, thus causing great harm to the health of the human body. For example, ochratoxin A (OTA) and fumonisin B1(FB1) can cause damage to human liver, and liver cancer can be caused by long-term effect. China stipulates that the content of OTA in grains and products thereof cannot exceed 5.0 mu g/kg, and the content of OTA in feed raw materials and products cannot exceed 100 mu g/kg. The European Union has the following regulation on the total content of fumonisins B1(FB1) and B2(FB2) in corn, and not more than 4mg/kg in infant corn products, and not more than 0.2mg/kg in infant corn products. Therefore, it is very important to realize the simultaneous and rapid detection of various trace toxins in food.
At present, the domestic and foreign detection methods for toxins and antibiotics in food include thin-layer chromatography, enzyme-linked immunosorbent assay, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. Although the thin-layer chromatography is simple, the thin-layer chromatography has the defects of low sensitivity, poor reproducibility and the like; the enzyme-linked immunosorbent assay has high sensitivity and strong specificity, but the enzyme stability is insufficient, so that false positive can easily occur in the detection of complex matrix samples; the high performance liquid chromatography and the liquid chromatography-mass spectrometry have the advantages of high sensitivity, good reproducibility and the like, and various target objects can be detected simultaneously by utilizing different retention times of all components.
The synthesis of the molecular engram polymer comprises the steps of complexing template molecules with functional monomers in a solution through non-covalent bond action, and gradually polymerizing the functional monomers around the template molecules under the action of a cross-linking agent and an initiator. After the polymerization is completed, the template molecule is removed by washing sufficiently so that the binding sites (i.e., cavities) can bind to a substance similar in size, shape and position to the functional groups of the template molecule. The molecular imprinting has the characteristics of good stability, simple preparation and the like, but the specific recognition capability is poor.
The aptamer is a segment of RNA or DNA containing 20-100 nucleotides and can be obtained by screening through an exponential enrichment ligand phylogeny technology, and the aptamer has specific targeting capacity comparable to that of antibodies and enzymes and can be specifically combined with small molecules, proteins and even cells. Meanwhile, the aptamer has the advantages that the antibody and the enzyme do not have: the method has the advantages of chemical synthesis, easy chemical modification, small molecular weight, small steric hindrance when being combined with a target object, wide range of target ligands and the like.
The complete sample analysis process includes sample collection, preparation, analysis, data processing and result reporting. Samples taken from the environment, food, clinical specimens, or biological fluids, often contain complex chemical components, and direct analysis may not accurately detect low levels of the target analyte. On the other hand, impurities in the sample may also form blockages, causing damage to the analysis instrument. Sample pretreatment is a key step in the whole analysis process, and directly influences the accuracy and precision of sample analysis. At present, various pretreatment technologies generally have a bipolarization development trend, namely the applicability of the pretreatment technologies is greatly reduced when high selectivity is pursued, and the selectivity of a sample pretreatment method is poor when the method applicability is pursued to achieve the purpose of high-throughput analysis. How to simultaneously realize high selectivity, high flux and customized detection of different samples has research value.
Disclosure of Invention
Based on the above, the invention aims to provide a preparation method of a nucleic acid aptamer molecular imprinting cooperative recognition stainless steel mesh, wherein a nucleic acid aptamer is added in a process of synthesizing a molecular imprinting polymer, and based on the cooperative recognition effect of the nucleic acid aptamer and the molecular imprinting polymer, the specific recognition capability of a ligand template molecule is improved through the cooperative recognition of the nucleic acid aptamer-template molecule-functional monomer, and meanwhile, the stability of the nucleic acid aptamer is enhanced through immobilizing the nucleic acid aptamer in a polymer skeleton structure, so that the advantages of two molecular recognition technologies of molecular imprinting and the nucleic acid aptamer are complementary, and a target object is recognized with high selectivity.
The technical scheme adopted by the invention is as follows:
a preparation method of a nucleic acid aptamer molecular imprinting synergistic recognition stainless steel mesh comprises the following steps:
(1) sequentially carrying out organic solvent soaking, alkali washing, deionized water washing and drying treatment on the stainless steel mesh;
(2) performing surface treatment on the stainless steel mesh obtained in the step (1) by using tannic acid;
(3) silanizing the stainless steel mesh obtained in the step (2) by using gamma-methacryloxypropyltrimethoxysilane;
(4) and (3) adding the stainless steel mesh, the template molecules and the aptamer obtained in the step (3) into a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, then sequentially adding a functional monomer, a cross-linking agent and an initiator to carry out polymerization reaction, and finally washing with an eluent to remove the template molecules, thereby obtaining the aptamer molecular imprinting synergistic recognition stainless steel mesh.
Different template molecules can be selected in the step (4) according to different targets, so that a plurality of corresponding aptamer molecular imprinting cooperative recognition stainless steel meshes (also called aptamer-molecular imprinting polymer coating filter discs) are prepared, and a plurality of filter discs with different specific recognition targets can be stacked and combined in a solid phase extraction cartridge according to analysis requirements, so that the customizable, high-flux and high-selectivity recognition of a plurality of toxins in a complex matrix is realized.
The aptamer molecular imprinting synergistic recognition stainless steel mesh prepared by the invention can be used for analyzing and detecting a plurality of toxin molecules in a complex matrix. Compared with the traditional molecularly imprinted polymer, the aptamer molecularly imprinted synergistic recognition stainless steel mesh has the following advantages: 1. the aptamer is added into the recognition system, and the selectivity of the material to a target object is greatly enhanced through the synergistic recognition effect of the molecular imprinting and the aptamer, so that the effect that one plus one is larger than two is achieved; 2. by replacing the added template molecules and the types of the nucleic acid aptamers, the nucleic acid aptamer molecular imprinting synergistic recognition stainless steel mesh which can specifically recognize different toxin molecules can be obtained, and can be used as a filler in a solid phase extraction column in a combined manner, so that the simultaneous recognition of various toxins with customization, high flux and high selectivity can be realized; 3. by immobilizing the aptamer in a polymer backbone structure, aptamer stability is enhanced.
In addition, the stainless steel mesh is selected as the base material to carry out molecular imprinting, and experiments prove that the method has feasibility and large contact area with an imprinting object. Meanwhile, the stainless steel mesh can be assembled and disassembled as a whole in the solid-phase extraction according to requirements, so that the subsequent free combination of one or more than two molecular imprinting materials in the solid-phase extraction column is convenient to realize, and the use is convenient.
Specifically, in the organic solvent soaking treatment in the step (1), the organic solvent is acetone or benzene, the soaking time is 50-100 hours, and the organic solvent is replaced every 20-30 hours.
Specifically, in the alkali washing treatment in the step (1), the adopted alkali liquor contains sodium hydroxide, sodium carbonate and sodium phosphate in a mass ratio of 5:2:2, and the alkali washing is carried out under the water bath heating condition of 70-90 ℃ for 20-40 minutes.
Specifically, the step (2) includes: and (2) soaking the stainless steel mesh obtained in the step (1) in a mixed solution prepared from tannic acid, ferric chloride and 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, performing surface treatment for 1-3 minutes at room temperature, taking out the stainless steel mesh, washing with deionized water and drying. The tannic acid and the ferric chloride can perform a complex reaction under the condition that the pH value is 7.45 so as to form a film on the substrate material, so that the surface of the stainless steel mesh is provided with hydroxyl. The reaction is carried out at room temperature, the oscillation is carried out for 1 to 3 minutes, and after the reaction is finished, the stainless steel mesh is taken out, washed by deionized water and dried.
Specifically, in the mixed solution in the step (2), the molar ratio of ferric chloride to tannic acid is 37: 24.
Specifically, the step (3) includes: and (3) adding the stainless steel mesh obtained in the step (2) into a silanization reagent prepared from gamma-methacryloxypropyltrimethoxysilane, methanol and water, and performing silanization treatment, so that hydroxyl groups obtained by chemical treatment on the stainless steel mesh can be converted into double bonds to participate in the polymerization of molecular imprinting. After the reaction, the stainless steel mesh was taken out, washed with methanol and dried.
Specifically, in the silylation reagent in the step (3), the volume ratio of the gamma-methacryloxypropyltrimethoxysilane to the methanol to the water is 1:1: 8.
Specifically, in the step (4), the functional monomer is methacrylic acid, the crosslinking agent is N, N' -methylenebisacryloyl, and the initiator is ammonium persulfate and tetramethylethylenediamine with a solution volume ratio of 17: 1.
The invention also provides the aptamer molecular imprinting synergistic recognition stainless steel mesh prepared by the preparation method, and the mesh can be applied to solid phase extraction.
The invention also provides a solid phase extraction column which comprises a filler consisting of the aptamer molecular imprinting cooperative recognition stainless steel mesh. The meshes for specifically recognizing different target objects are combined in the solid phase extraction column, so that high-throughput, high-selectivity and customized detection of various toxins can be realized.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of a preparation method of a aptamer molecular imprinting cooperative recognition stainless steel mesh;
FIG. 2 is a scanning electron microscope and energy spectrum characterization element diagram of aptamer molecular imprinting synergistic recognition of a stainless steel mesh;
FIG. 3 is a confocal laser imaging diagram of aptamer molecular imprinting cooperative identification of a stainless steel mesh;
FIG. 4 is a graph showing the results of optimizing the amounts of initiators APS and TMEDA;
FIG. 5 shows the result of optimizing the amount of MBA used as a crosslinking agent;
FIG. 6 is a graph comparing the OTA extraction effect of different imprinted stainless steel meshes;
FIG. 7 is a graph comparing the extraction effect of different stainless steel mesh sheets on FB 1;
FIG. 8 is a graph showing the effect of OTA aptamer molecular imprinting on the synergistic recognition of aptamer stability in stainless steel mesh;
FIG. 9 is a graph of the effect of stability of OTA aptamers chemically bonded directly to the surface of a stainless steel mesh;
FIG. 10 is a graph showing the effect of molecular imprinting of FB1 aptamer on the stability of the aptamer in cooperative recognition in a stainless steel mesh;
FIG. 11 is a graph showing the effect of the stability of an FB1 aptamer chemically bonded directly to the surface of a stainless steel mesh;
FIG. 12 is a graph showing the effect of the aptamer molecular imprinting on the selectivity of the cooperative recognition of stainless steel mesh.
Detailed Description
Referring to fig. 1, the preparation method of the aptamer molecular imprinting cooperative recognition stainless steel mesh according to the present invention includes the following steps:
(1) sequentially carrying out organic solvent soaking, alkali washing, deionized water washing and drying treatment on the stainless steel mesh;
(2) performing surface treatment on the stainless steel mesh obtained in the step (1) by using tannic acid;
(3) silanizing the stainless steel mesh sheet obtained in the step (2) with gamma-methacryloxypropyltrimethoxysilane (KH 570);
(4) and (3) adding the stainless steel mesh, the template molecules and the aptamer obtained in the step (3) into a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution, then sequentially adding a functional monomer, a cross-linking agent and an initiator to carry out polymerization reaction, and finally washing with an eluent to remove the template molecules to obtain the aptamer molecular imprinting synergistic recognition stainless steel mesh.
By changing the types of the template molecules and the aptamers, the aptamer molecular imprinting cooperative recognition stainless steel mesh for specifically recognizing different targets can be obtained.
Specifically, in the organic solvent soaking treatment in the step (1), the organic solvent is acetone or benzene, the soaking time is 50-100 hours, and the organic solvent is replaced every 20-30 hours. In the alkali washing treatment in the step (1), the adopted alkali liquor contains sodium hydroxide (NaOH) and sodium carbonate (Na) in a mass ratio of 5:2:22CO3) And sodium phosphate (Na)3PO4) The alkali washing is carried out under the heating condition of water bath at 70-90 ℃, and the alkali washing time is 20-40 minutes.
The step (2) is specifically as follows: soaking the stainless steel mesh sheet obtained in the step (1) in tannin and ferric chloride (FeCl)3) Performing surface treatment for 1-3 minutes at room temperature in a mixed solution prepared from HEPES buffer solution, taking out the stainless steel mesh, washing with deionized water and drying; in the mixed solution, FeCl3The molar ratio to tannic acid was 37: 24.
The step (3) is specifically as follows: adding the stainless steel mesh obtained in the step (2) into a silanization reagent prepared from KH570, methanol and water, performing silanization treatment, taking out the stainless steel mesh after the silanization treatment, washing with methanol and drying; in the silylation reagent, the volume ratio of KH570 to methanol to water is 1:1: 8.
In the step (4), the functional monomer is methacrylic acid (MAA), the crosslinking agent is N, N' -Methylenebisacryloyl (MBA), the initiator is Ammonium Persulfate (APS) and Tetramethylethylenediamine (TMEDA), and preferably, the volume ratio of APS to TMEDA is 17: 1; the polymerization reaction is carried out at 60 ℃ under oscillation;
and (3) arranging and stacking the prepared nucleic acid aptamer molecular imprinting cooperative recognition stainless steel meshes, and filling the stainless steel meshes serving as fillers into a tube body of a solid-phase extraction column to obtain the solid-phase extraction column shown in figure 1.
According to the invention, different template molecules can be selected based on different types of analysis objects, a plurality of corresponding aptamer molecular imprinting cooperative identification stainless steel mesh sheets (also called aptamer-molecular imprinting polymer coating filter sheets) are respectively prepared according to the preparation method, and then a plurality of filter sheets are stacked and combined in a solid phase extraction small column according to analysis requirements, so that the solid phase extraction device shown in figure 1 is obtained, and thus, the customizable, high-flux and high-selectivity identification of a plurality of toxins in a complex matrix is realized.
Example 1
In the embodiment, ochratoxin A (OTA) is selected as a template molecule, and the OTA aptamer molecular imprinting cooperative identification stainless steel mesh is prepared according to the following steps:
adding the stainless steel mesh and acetone into the beaker, soaking the stainless steel mesh for 72 hours by using the acetone, and replacing the acetone once every 24 hours. The preparation contains 50g/L NaOH and 20g/L Na2CO3、20g/L Na3PO4And (3) adding the stainless steel mesh sheet soaked by the acetone into the mixed alkali liquor, washing for 30min under the condition of stirring and heating in a water bath at the temperature of 80 ℃, taking out the stainless steel mesh sheet, washing with deionized water, and drying for later use. Then, 5mL of HEPES buffer solution having a pH of 7.45, a clean stainless steel mesh sheet, and 37. mu.L of 50mM FeCl were sequentially added to a clean reaction vessel3And (3) oscillating the solution and 24 mu L of 50mM tannic acid solution at room temperature for 1min, taking out the stainless steel mesh after the reaction is finished, washing the stainless steel mesh with deionized water, and drying the stainless steel mesh. Then, in another clean reaction vessel, the stainless steel mesh treated by tannic acid, 10mL KH570, 10mL methanol and 80mL deionized water are added, the reaction vessel is placed in a shaking table, the reaction is carried out for 30min under the conditions that the temperature is 30 ℃ and the rotation speed of the shaking table is 250rmp, and after the reaction is finished, the stainless steel mesh is washed clean by methanol and dried for standby.
Adding 3mL of HEPES buffer solution with the pH value of 7.45, the treated stainless steel mesh, 10 muL of 1mg/mL OTA solution, 50 muL of 66 mug/mL OTA aptamer solution and 25 muL of MAA into a 10mL centrifuge tube, filling nitrogen into the centrifuge tube, placing the centrifuge tube into a shaking table, pre-reacting for 20min at the temperature of 60 ℃ and the rotation speed of the shaking table of 250rmp, adding 36.0mg of MBA cross-linking agent, filling nitrogen into the centrifuge tube, placing the centrifuge tube into the shaking table, pre-polymerizing for 30min at the temperature of 60 ℃ and the rotation speed of the shaking table of 250rmp, finally adding 34 muL of 0.2499mol/L APS solution and 2 muL of 6.6695mol/L TMDA solution, refilling nitrogen into the centrifuge tube, placing the centrifuge tube into the shaking table, and polymerizing for 20h at the temperature of 60 ℃ and the rotation speed of the shaking table of 250 rmp. After the reaction is finished, the stainless steel mesh is washed by methanol (v/v) containing 10% acetic acid for multiple times to remove the template molecules, and the OTA aptamer molecular imprinting synergistic recognition stainless steel mesh is obtained.
The obtained OTA aptamer molecular imprinting synergistic recognition stainless steel mesh is stacked in a combined mode and filled in a tube body of a solid phase extraction column as a filler, and the obtained solid phase extraction column is used for detecting OTA toxin in an actual sample.
Example 2
In this example, fumonisin B1(FB1) was selected as a template molecule, and FB1 aptamer molecular imprinting cooperative recognition stainless steel mesh was prepared as follows:
adding the stainless steel mesh and acetone into the beaker, soaking the stainless steel mesh for 72 hours by using the acetone, and replacing the acetone once every 24 hours. The preparation contains 50g/L NaOH and 20g/L Na2CO3、20g/L Na3PO4And (3) adding the stainless steel mesh sheet soaked by the acetone into the mixed alkali liquor, washing for 30min under the condition of stirring and heating in a water bath at the temperature of 80 ℃, taking out the stainless steel mesh sheet, washing with deionized water, and drying for later use. Then, 5mL of HEPES buffer solution having a pH of 7.45, a clean stainless steel mesh sheet, and 37. mu.L of 50mM FeCl were sequentially added to a clean reaction vessel3And (3) oscillating the solution and 24 mu L of 50mM tannic acid solution at room temperature for 1min, taking out the stainless steel mesh after the reaction is finished, washing the stainless steel mesh with deionized water, and drying the stainless steel mesh. Then, in another clean reaction vessel, the stainless steel mesh treated by tannic acid, 10mL KH570, 10mL methanol and 80mL deionized water are added, the reaction vessel is placed in a shaking table, the reaction is carried out for 30min under the conditions that the temperature is 30 ℃ and the rotation speed of the shaking table is 250rmp, and after the reaction is finished, the stainless steel mesh is washed clean by methanol and dried for standby.
Adding 3mL of HEPES buffer solution with the pH value of 7.45, the treated stainless steel mesh, 10 mu L of 1mg/mL FB1 solution, 50 mu L of 66 mu g/mL FB1 aptamer solution and 25 mu L of MAA into a 10mL centrifuge tube, filling nitrogen into the centrifuge tube, placing the centrifuge tube into a shaking table, pre-reacting for 20min at the temperature of 60 ℃ and the rotation speed of the shaking table of 250rmp, adding 36.0mg of MBA of cross-linking agent, filling nitrogen into the centrifuge tube, placing the centrifuge tube into the shaking table, pre-polymerizing for 30min at the temperature of 60 ℃ and the rotation speed of the shaking table of 250rmp, finally adding 34 mu L of 0.2499mol/L APS solution of initiator and 2 mu L of 6.6695mol/L TMDA solution, refilling nitrogen into the centrifuge tube, placing the centrifuge tube into the shaking table, and polymerizing for 8h at the temperature of 60 ℃ and the rotation speed of the shaking table of 250 rmp. After the reaction is finished, the stainless steel mesh is washed for multiple times by methanol (v/v) containing 10% acetic acid to remove the template molecules, and the FB1 aptamer molecular imprinting cooperative recognition stainless steel mesh is obtained.
The obtained FB1 aptamer molecular imprinting is cooperated with recognition stainless steel mesh sheets to be stacked in a combined mode and used as filler to be filled in a tube body of a solid phase extraction column, and the obtained solid phase extraction column is used for detecting FB1 toxin in an actual sample.
The invention also combines two aptamer molecular imprinting stainless steel mesh sheets of OTA and FB1 prepared in examples 1 and 2 respectively for use in separating and detecting OTA and FB1 in mildewed grapes, corns, feeds and the like.
Characterization and test results
Referring to FIG. 2, which is a scanning electron microscope and energy spectrum characterization element diagram of the aptamer molecular imprinting synergistic recognition stainless steel mesh prepared by the present invention, it can be seen from the element energy spectrum that Fe, Cr, and Ni elements are the main elements contained in the stainless steel mesh substrate material, i.e., the distribution positions of Fe, Cr, and Ni elements correspond to the positions of the stainless steel mesh substrate material; C. n, O, the distribution position of the element corresponds to the position of the molecular imprinting coating formed by the preparation method; as can be seen from the figure, the distribution positions of all elements are overlapped, which shows that the position of the molecular imprinting coating formed on the stainless steel mesh by the preparation method of the invention is overlapped with the position of the stainless steel mesh substrate material, and shows that the molecular imprinting coating is uniformly distributed on the surface of the stainless steel mesh.
Please refer to fig. 3, which is a 3D image of confocal laser imaging of aptamer molecular imprinting cooperative recognition stainless steel mesh prepared according to the present invention. The aptamer marked by the fluorescent group is used in the preparation method of the invention, the aptamer molecular imprinting with the fluorescent group is synthesized to cooperatively identify the stainless steel mesh, the stainless steel mesh is excited by the excitation wavelength corresponding to the fluorescent group under a laser confocal microscope, and the fluorescence is uniformly distributed on the stainless steel mesh according to the obtained 3D confocal image, which indicates that the aptamer is uniformly distributed on the surface of the whole stainless steel mesh.
Referring to fig. 4 and fig. 5, the optimization results of the amounts of the initiator APS and TMEDA and the crosslinking agent MBA in the preparation method of the aptamer molecular imprinting cooperative recognition stainless steel mesh are respectively shown. The thickness of the molecular imprinting coating formed on the stainless steel mesh is represented by the weight percentage of the carbon element, and the result of fig. 4 shows that when the dosage of the initiator APS is 34 μ L and the dosage of the TMEDA is 2 μ L, the percentage of the carbon element in the molecular imprinting coating is the highest, which indicates that the optimal dosages of the initiator APS and the TMEDA are 34 μ L and 2 μ L respectively; the results in fig. 5 show that when the amount of the crosslinking agent MBA is 36.0mg, the percentage of carbon in the molecularly imprinted coating is the highest, which indicates that the optimal amount of the crosslinking agent MBA is 36.0mg, and the thickness of the molecularly imprinted coating synthesized under the optimal conditions is moderate.
Referring to fig. 6, which is a graph comparing the extraction effect of different imprinted stainless steel meshes on OTA, wherein MIP-Apt is the synthesized OTA aptamer molecular imprinting synergistic recognition stainless steel mesh of example 1; MIP is an imprinted mesh which is synthesized only by molecular imprinting and not adding a nucleic acid aptamer; apt is a blot mesh which is only added with a nucleic acid aptamer and is not added with an MAA functional monomer; NIP is a blank imprinted mesh added with disordered OTA aptamer and MAA and without template molecules. As can be seen from fig. 6, the extraction amount of the MIP-Apt imprinted mesh for OTA is greater than the sum of the extraction amounts of the MIP imprinted mesh and the Apt imprinted mesh for OTA, and is significantly greater than the extraction amount of the NIP imprinted mesh, which indicates that the MIP-Apt imprinted mesh synthesized according to the present invention achieves the effect of synergistic recognition of molecular imprinting and aptamer, and greatly enhances the selectivity of the imprinted mesh for OTA.
Referring to FIG. 7, which is a graph comparing the extraction effect of different imprinted stainless steel meshes on FB1, MIP-Apt is the FB1 aptamer synthesized in example 1 with molecular imprinting for synergistic recognition of stainless steel meshes; MIP is an imprinted mesh which is synthesized only by molecular imprinting and not adding a nucleic acid aptamer; apt is a blot mesh which is only added with a nucleic acid aptamer and is not added with an MAA functional monomer; NIP is a blank imprinted mesh added with disordered OTA aptamer and MAA and without template molecules. As can be seen from fig. 7, the extraction amount of the MIP-Apt imprinted mesh to FB1 is greater than the sum of the extraction amounts of the MIP imprinted mesh and the Apt imprinted mesh to FB1, and is significantly greater than the extraction amount of the NIP imprinted mesh, which indicates that the MIP-Apt imprinted mesh synthesized according to the present invention realizes the effect of synergistic recognition of molecular imprinting and a nucleic acid aptamer, and greatly enhances the selectivity of the imprinted mesh to FB 1.
Referring to fig. 8 and 9, fig. 8 is a graph showing the comparison of the amount of OTA extracted before and after the MIP-Apt stainless steel mesh synthesized in example 1 is subjected to enzymatic hydrolysis; FIG. 9 shows a comparison of the amount of OTA extracted before and after the same enzymatic digestion of aptamers chemically bound directly to the surface of a stainless steel mesh. As can be seen from FIG. 9, the aptamer is directly bonded on the surface of the stainless steel mesh, and the amount of the aptamer extracted from OTA by the stainless steel mesh is significantly reduced by enzymolysis, which indicates that part of the aptamer is enzymolyzed, thereby reducing the amount of the mesh extracted from OTA. As can be seen from the extract amount change chart in FIG. 8, the MIP-Apt mesh prepared by the method of the invention has good extraction performance under the same enzymolysis, and because the aptamer is coated in the molecularly imprinted polymer skeleton, the molecularly imprinted polymer skeleton protects the aptamer and inhibits the enzymolysis of the aptamer.
Referring to FIGS. 10 and 11, FIG. 10 is a graph showing the comparison of the amount of FB1 extracted before and after the enzymatic hydrolysis of the MIP-Apt stainless steel mesh sheet synthesized in example 2; FIG. 11 is a graph showing a comparison of the amount of FB1 extracted from the stainless steel mesh before and after the same enzymatic digestion, in which the aptamer was chemically bonded directly to the surface of the stainless steel mesh. As can be seen from FIG. 11, the aptamer is directly bonded on the surface of the stainless steel mesh, and the amount of FB1 extracted by the stainless steel mesh is significantly reduced by the enzymolysis, which indicates that part of the aptamer is subjected to enzymolysis, thereby reducing the amount of FB1 extracted by the mesh. As can be seen from the extract amount change diagram in FIG. 10, the MIP-Apt mesh prepared by the method of the present invention has good extraction performance under the same enzymolysis, and because the aptamer is coated in the molecularly imprinted polymer skeleton, the molecularly imprinted polymer skeleton protects the aptamer, thereby inhibiting the enzymolysis of the aptamer.
Referring to FIG. 12, which is a graph showing the selective effect of aptamer molecular imprinting in synergistic recognition of stainless steel mesh, the OTA MIP-Apt imprinted mesh synthesized in example 1, and a control group of OTA MIP mesh, OTA Apt mesh and OTA NIP mesh, and FB1MIP-Apt blotting meshes synthesized in example 2, and FB MIP meshes, FB Apt meshes, and FBNIP meshes of the control group, five toxins including OTA, FB1, Zearalenone (ZEN), aflatoxin G2(AFG2) and aflatoxin G2(AFB2) are separated and detected, so that the graph shows that the OTA MIP-Apt blotting mesh has good selectivity on OTA, the FB1MIP-Apt blotting mesh has good selectivity on FB1, and experimental results show that the interference of other three toxins ZEN, AFG2 and AFB2 on nucleic acid aptamer molecular blotting synergistic recognition stainless steel mesh separation detection target is small.
The aptamer molecular imprinting synergistic recognition stainless steel mesh prepared by the invention can be used for analyzing and detecting a plurality of toxin molecules in a complex matrix. Compared with the traditional molecularly imprinted polymer, the aptamer molecularly imprinted synergistic recognition stainless steel mesh has the following advantages: 1. the aptamer is added into the recognition system, and the selectivity of the material to a target object is greatly enhanced through the synergistic recognition effect of the molecular imprinting and the aptamer, so that the effect that one plus one is larger than two is achieved; 2. by replacing the added template molecules and the types of the nucleic acid aptamers, the nucleic acid aptamer molecular imprinting synergistic recognition stainless steel mesh which can specifically recognize different toxin molecules can be obtained, and can be used as a filler in a solid phase extraction column in a combined manner, so that the simultaneous recognition of various toxins with customization, high flux and high selectivity can be realized; 3. by immobilizing the aptamer in a polymer backbone structure, aptamer stability is enhanced.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. A preparation method of a aptamer molecular imprinting synergistic recognition stainless steel mesh is characterized by comprising the following steps:
(1) sequentially carrying out organic solvent soaking, alkali washing, deionized water washing and drying treatment on the stainless steel mesh;
(2) performing surface treatment on the stainless steel mesh obtained in the step (1) by using tannic acid;
(3) silanizing the stainless steel mesh obtained in the step (2) by using gamma-methacryloxypropyltrimethoxysilane;
(4) and (3) adding the stainless steel mesh, the template molecules and the aptamer obtained in the step (3) into a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, then sequentially adding a functional monomer, a cross-linking agent and an initiator to carry out polymerization reaction, and finally washing with an eluent to remove the template molecules, thereby obtaining the aptamer molecular imprinting synergistic recognition stainless steel mesh.
2. The method of claim 1, wherein: in the organic solvent soaking treatment in the step (1), the organic solvent is acetone or benzene, the soaking time is 50-100 hours, and the organic solvent is replaced once every 20-30 hours.
3. The method of claim 1, wherein: in the alkali washing treatment in the step (1), alkali liquor contains sodium hydroxide, sodium carbonate and sodium phosphate in a mass ratio of 5:2:2, and alkali washing is carried out under the water bath heating condition of 70-90 ℃ for 20-40 minutes.
4. The method of claim 1, wherein: the step (2) comprises the following steps: and (2) soaking the stainless steel mesh obtained in the step (1) in a mixed solution prepared from tannic acid, ferric chloride and 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, performing surface treatment for 1-3 minutes at room temperature, taking out the stainless steel mesh, washing with deionized water and drying.
5. The method of claim 4, wherein: in the mixed solution in the step (2), the molar ratio of ferric chloride to tannic acid is 37: 24.
6. The method of claim 1, wherein: the step (3) comprises the following steps: and (3) adding the stainless steel mesh obtained in the step (2) into a silanization reagent prepared from gamma-methacryloxypropyltrimethoxysilane, methanol and water, performing silanization treatment, taking out the stainless steel mesh after the silanization treatment, washing with methanol and drying.
7. The method of claim 6, wherein: in the silylation reagent in the step (3), the volume ratio of the gamma-methacryloxypropyltrimethoxysilane to the methanol to the water is 1:1: 8.
8. The method of claim 1, wherein: in the step (4), the functional monomer is methacrylic acid, the cross-linking agent is N, N' -methylene diacryloyl, and the initiator is ammonium persulfate and tetramethylethylenediamine with a solution volume ratio of 17: 1.
9. The aptamer molecular imprinting synergistic recognition stainless steel mesh prepared by the preparation method of any one of claims 1 to 8.
10. A solid phase extraction column, characterized in that: comprising a filler consisting of the aptamer molecular imprinting cooperative recognition stainless steel mesh of claim 9.
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